Process for producing prostaglandins

ABSTRACT

1. A PROCESS FOR THE BIOSYNTHESIS OF PROSTAGLANDINS, COMPRISING THE STEPS OF (A9 GROWING THE BACTERIUM PSEUDOMONAS AERUGINOSA IN THE PRESENCE OF OXYGEN IN A CULTURE MEDIUM ESSENTIALLY FREE OF FATTY ACIDS AND CONTAINING AVAILABLE NITROGEN AND CARBON THEREBY TO FOM PROSTAGLANDINS; AND (B) ISOLATING THE PRODUCT PROSTAGLANDING THUS FORMED.

US. 'Cl. 19529 I United States Patent ABSTRACT OF THE DISCLOSURE Postaglandins and/or prostaglandin-like compounds are synthesized by a microbiological process using Pseudomonas aeruginosa in a nitrogen-containing, fatty acid-free culture medium.

This invention relates to a class of biologically active lipids and more particularly to a process for the biosynthesis of a class of lipids referred to as prostaglandins.

In recent years a group of unsaturated carboxylic acids containing 20 carbon atoms, in which C-8 to C-12 compose a five-membered ring, have been identified as existing in biological systems. These compounds have been given the common generic name prostaglandins and they have been the subject of intensive research with respect to their various roles in biological systems, their use as pharmacologically active agents in treating certain conditions, and their use as precursors in the formation of new drugs.

Although there are a number of prostaglandins known, the process for their production which is the subject of this invention is concerned with so-called prostaglandins (A, E and F series), each of which has characteristic substituents on the carbon chains which vary according to their fatty acid precursors, and with prostaglandinlike materials.

Thus the prostaglandins designated A have a characterizing ket grouping on C-9 and three possible structures for prostaglandinA (PGA) may be represented as PGA1 ii WA PGAz PGA;

depending upon whether the fatty acid precursor is 8,11, l4-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid or 5,8,11,14,17-eicosapentaenoic acid. In similar fashion, prostaglandins designated E have a characterizing. keto grouping on the C-9 and a hydroxyl grouping on the 0-11 and may be represented by the following structural formulas OOOH PGE3

Prostaglandins designated F, have characterizing hydroxyl groupings on the C-9 and C-11 and may be represented as having the following structural formulas:

OH C0011 The prostaglandins may be isolated from biological materials, and a l6-step synthesis leading to PGF has been reported (see Total Synthesis of Prostaglandins by Elias J. Corey, Proc. Robert A. Welch Found. C any. Chem.

Res. (Pub. 1969) 12, 51-79). The conversion of prostaglandins derived from the coral Plexaura homomwlla to thesis of prostaglandins in which unsaturated fatty acids,

having a 1,4diene group in their chains, are subjected to the oxygenating activity of a species of Subphylum 2 of Phylum III. US. Pat. 3,644,502 discloses a process for chemically synthesizing prostaglandins from o-methoxyphenylacctic acid.

There is, however, a need for an improved process for producing the prostaglandins and prostaglandin-like materials. Such an improved process should desirably be less complicated and time-consuming than those now employed and should make it possible to provide such materials at lower cost.

Since prostaglandins occur endogenously in animals including humans, they find one very important use in studying the inhibitions of these naturally-occurring materials by such antagonists as polyphloretin phosphate, dibenzoxazepine hydrazine derivatives, the active ingredients in aspirin, etc. In general, these compounds are known to have hypotensive and smooth muscle-stimulating activity. Prostaglandins have been used in performing therapeutic abortions in several countries, and their use has been endorsed by the Committee on the Safety of Drugs in Great Britain. They have also been found to have a bronchodilating effect in the treatment of asthmatics and they are known to have a regulating effect on blood pressure and sodium excretion. (See for example Science Volume 171, pp. 502-504 (1971).)

It is therefore a primary object of this invention to provide an improved process for producing prostaglandins and prostaglandin-like substances. It is another object of this invention to provide a process of the character described which is relatively simple to manipulate and which may make it possible to produce prostaglandins and prostaglandin-like substances econorm'cally. Other objects of the invention will in part be obvious and will in part be apparent hereinafter.

According to the process of this invention, prostaglandins and prostaglandin-like substances are synthesized by strains of the bacterium Pseudomonas aeruginosa grown in nitrogenand carbon-containing culture media in the presence of oxygen.

The culture medium must be one which provides nitrogen and carbon in usable forms. Exemplary of such media are a broth containing a hydrolyzed or partially hydrolyzed protein such as soya protein as the nitrogen source and dextrose as the carbon source and a chemically defined broth containing many of amino acids known to form proteins serving as both the nitrogen and carbon sources. The culture medium may be enriched with heat-inactivated human serum, rabbit serum albumin, etc., serving as additional protein sources. The amount of such protein additive or additives should preferably be no greater than about 4% by weight of the growth medium.

The temperature of the culture during the production of the prostaglandins and prostaglandin-like substances may range between about 4 C. and 40 C., the higher temperatures being preferred inasmuch as the production rate is materially increased with increasing temperature.

The pH of the culture medium just prior to inoculation with Pseudomonas aeruginosa should be about neutral, and the formation of the prostaglandin product may take place within a pH range of from about 5 to 9.

The prostaglandin product is extracted from the culture medium with ethyl acetate or other appropriate solvents and it is identified by radioimmunoassay techniques.

The following examples, which are meant to be ill-ustrative and not limiting, are provided to further describe the process of this invention.

Pseudomonas aeruginosa was inoculated into 1.6 ml. trypticase soy broth (TSB), free of any added fatty acids, and the cultures incubated at 37 C., 23 C. and 4 C. Samples of 0.3 ml. were taken at varioustime intervals, diluted with saline, centrifuged and the supernatant fluids measured for F prostaglandin content. In this example, as well as in all of the following examples, the prostaglandins 4 were identified through the use of the radioimmunoassay technique of Stylos et a1. (see Intra-Science Chem. Rept., Volume 6, Number 1 (1972) pp. 67-71) with the following modifications. To increase sensitivity of the assay 0.1 ml. of H-F containing approximately 7500 d.p.m. (-10 pg.) was used instead of HF;;,,. Bound prostagl'andins were separated from unbound by centrifugation of 2nd antibody precipitates. The supernatants were decanted and the tubes were inverted onto paper towels to drain 10 minutes and any remaining fluid removed by suction. The precipitates were dissolved in 0.2 ml. of 0.1 N NaOH and decanted into vials containing 7 ml. of scintillation fluid. The tubes were rinsed twice with 1.5 ml. of fluid and these rinses added to the scintillation vials for counting. The PGF immunoassay according to this modified technique has been twice validated in double blind experiments with GLC/ mass spectrometry data. The prostaglandins (or prostaglandin-like) compounds were also identified by thin layer partition chromatography.

The production of prostaglandin and prostaglandin-like substances using a series of such cultures is given below in Table 1.

TABLE 1 Prostaglandin production by P5. ueruginosa using trypticase soy broth Growth time Days Weeks Production e Temperature, 0.:

To determine the oxygen requirement for synthesis of the prostaglandin product, cultures were incubated in glass tubes with cotton plugs to allow free air exchange and in screw cap vials to obtain microaerop'hilic conditions.

Psudomonas aeruginosa was inoculated into 1.8 ml. TSB or 1.8 ml. TSB containing 1 ig/ml. arachidonic acid and all cultures incubated at 37 C. Samples of 0.3 ml. were removed at various time intervals, and treated as described for the above examples. The quantities of prostaglandin product resulting under these conditions are given in Table 2.

TABLE 2 Prostaglandln production under varying aerobic conditions [In ng./ml.]

Aerobic conditions 0 day 1 day 2 days Remarks With air 0. 00 0. 85 L 41 Heavy growth of organism. I With air plus araehidonie 0. 00 0. 75 0. 95 Do.

801 Microaerophilie 0. 00 0. 00 0. 00 Do.

I conditions used to obtain the data in Table 2. The bacteria used were Salmonella emeriditis, Salmonella typhimuriwm (2 strains), Escherichia coli, Salmonella St. Paul, Salmonella typhosa, a group D Salmonella and a Staphylococcus. None of these produced a prostaglandin or prostaglandinlike substance in either TSB or in TSB supplemented with arachidonic acid or with human serum.

The trypticase soy broth was enriched with varying amounts of heat-inactivated human serum to determine the effect of serum components on prostaglandin biosynthesis. Tubes containing 1.7 ml. of enriched broth were incubated at 37 C. and 0.3 ml. samples were removed after 1, 3 and 5 days. Samples were diluted with 0.9 ml. saline and treated as described above. The quantities of prostaglandin production are given in Table 3.

Figures for 100% TSB taken from an earlier example for comparison.

The inclusion of up to about 50% by weight of the heat-inactivated human serum generally increased the production of the prostaglandin product in the longer reaction runs.

For more rigorous identification of the prostaglandins and prostaglandin-like substances produced by the process of this invention, Pseudomonas aerugz'nosa culture supernatants were extracted by ethyl acetate and separated by thin layer chromatography. A flask containing 100 ml. TSB was inoculated and incubated for 4 days at 37 C. (the pH changed from 7.3 to 8.65 during this time). The supernatant was brought to pH 4.0 with HCl and extracted with 200 ml. ethyl acetate. The aqueous phase was removed and the ethyl acetate phase washed twice with distilled water and dried over sodium sulfate. The clarified ethyl acetate phase was taken to dryness under N solubilized and chromatographed using a solvent system which separated the A, E and F prostaglandin species: ethyl acetate-acetone-acetic acid (95 :5:1). Zones migrating with tritiated standards of A, E and F prostaglandins were eluted and measured in the radioimmunoassay. The amount of prostaglandins found in the three PG zone eluates were as follows: PGF, 8.7 ng.; PGE, 3.1 ng.; PGA, 16.2 ng. Thus the process of this invention is capable of producing prostaglandins and prostaglandin-like substances of the A, E and P series.

A chemically-defined broth, designated Bacto-Synthetic Broth AOAS by its supplier Difco Co., and prepared according to the formula given in J. AOCA, 47: 176 1964), was substituted for the trypticase soy broth used in the previous examples. This synthetic culture medium contained some eighteen amino acids; but it did not contain any fatty acids, which, if present, could be considered as precursors for prostaglandins or prostaglandin-like mate rials.

Pseudomonas aeruginosa was grown in the chemicallydefined medium under a number of diiferent conditions as summarized below in Table 4. The resulting culture supernatants were separated by centrifugation and measured for prostaglandin content by radioimmunoassay. The amounts of prostaglandins formed are given in Table 4.

TABLE 4 Postaglandin production by P3. acruginosa using chemically-defined culture medium Growth conditions in medium Production (ng./ml.)

Final Days 0. Additive PGF PGE IGA pH 1 37 None 0.4. 0.5 3.3 8.2 3 37 2 D phosphate 0.3 0.4 2.7 7.8

er. 3 37 5X amino acetic 0.3 0. 4 3.0 8. 3

ac 2; 3X glucose 0. 4. 0. 4 3. 7 7. 9 22 None 3 1. 0 17. 5 7. 9 37 do 1. 6 26. 8 8. 2 23 1 All values corrected for medium background.

2 Initial pH of chemically-defined medium was 7.1. 3 Single run using decreasing temperatures.

From these results it will be seen that additional amino acetic acid (glycine), glucose or phosphate butler did not appreciably alter the yields. The principal prostaglandin biosynthesized was PGA, while lesser amounts of PGE and PGF were formed.

It is evident from these examples that the process of this invention provides a simple synthetic route for the production of prostaglandins and prostaglandin-like materials; and it offers the possibility for large-scale economic synthesis of these materials.

It will thus be seen that the objects set forth above, among those made apparent from the preceding description are efficiently attained and, since certain changes may be made in carrying out the above process without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

We claim:

1. A process for the biosynthesis of prostaglandins, comprising the steps of (a) growing the bacterium Pseudomonas aeruginosa in the presence of oxygen in a culture medium essentially free of fatty acids and containing available nitrogen and carbon therby to form prostaglandins; and

(b) isolating the product prostaglandins thus formed.

2. A process in accordance with claim 1 wherein said culture medium is trypticase soy broth and said nitrogen is present in the form of protein naturally present in said soy broth.

3. A process in accordance with claim 2 wherein said trypticase soy broth contains an additional source of protein.

4. A process in accordance with claim 2 wherein said additional source of protein is heat-inactivated human serum.

5. A process in accordance with claim 1 wherein said culture medium is a chemically-defined medium containing amino acids which serve as said source of nitrogen.

6. A process in accordance with claim 1 wherein said available carbon is provided in the form of dextrose.

7. A process in accordance with claim 1 wherein said growing step is carried out between about 4 C. and 40 C.

8. A process in accordance with claim 1 wherein the growing of said bacterium is carried out at a pH between about 5 and 9.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, Primary Examiner U.S. Cl. X.R. --47, 96 

1. A PROCESS FOR THE BIOSYNTHESIS OF PROSTAGLANDINS, COMPRISING THE STEPS OF (A9 GROWING THE BACTERIUM PSEUDOMONAS AERUGINOSA IN THE PRESENCE OF OXYGEN IN A CULTURE MEDIUM ESSENTIALLY FREE OF FATTY ACIDS AND CONTAINING AVAILABLE NITROGEN AND CARBON THEREBY TO FOM PROSTAGLANDINS; AND (B) ISOLATING THE PRODUCT PROSTAGLANDING THUS FORMED. 